The present invention is directed to improvements in microbial fermentation methods for the production of ethanol from a gaseous substrate containing at least one reducing gas using anaerobic (or facultative) acetogenic bacteria.
Methods for producing ethanol, among other organic acids, alcohols, hydrogen and organic acid salts, from the microbial fermentation of gaseous substrates in media containing suitable nutrients and trace minerals using certain anaerobic bacteria have been disclosed by these inventors. For example, the inventors have previously disclosed that dilute gas mixtures are introduced into a bioreactor containing one or more strains of anaerobic bacteria that utilize the waste gas components by a direct pathway to produce a desired compound. The compound is recovered from the aqueous phase in a separate vessel or vessels, utilizing a suitable recovery method for the compound produced. Examples of recovery methods include extraction, distillation or combinations thereof, or other efficient recovery methods. The bacteria can be removed from the aqueous phase and recycled to the bioreactor to maintain high cell concentrations, thus maximizing productivity. Cell separation, if desired, is accomplished by centrifugation, membranous filtration, or other techniques. See, for example, International Patent Publication No. WO98/00558, published Jan. 8, 1998; U.S. Pat. No. 5,807,722; U.S. Pat. No. 5,593,886 and U.S. Pat. No. 5,821,111.
In addition to its major product, acetic acid, strains of the anaerobic bacterium Clostridium ljungdahlii are able to also produce ethanol as a product in the conversion of carbon monoxide (CO), hydrogen (H2) and carbon dioxide (CO2). The production of acetic acid (CH3COOH) and ethanol (C2H5OH) from CO, CO2 and H2 are shown by the following overall stoichiometric equations:4CO+2H2O→CH3COOH+2CO2  (1)4H2+2CO2→CH3COOH+2H2O  (2)6CO+3H2O→C2H5OH+4CO2  (3)6H2+2CO2→C2H5OH+3H2O  (4)
Several exemplary strains of C. ljungdahlii include strain PETC (U.S. Pat. No. 5,173,429); strain ERI-2 (U.S. Pat. No. 5,593,886) and strains C-01 and O-52 (U.S. Pat. No. 6,136,577). These strains are each deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, under Accession Nos.: 55383 (formerly ATCC No. 49587), 55380, 55988, and 55989 respectively. Each of the strains of C. ljungdahlii is an anaerobic, gram-positive bacterium with a guanine and cytosine (G+C) nucleotide content of about 22 mole %. These bacteria use a variety of substrates for growth, but not methanol or lactate. These strains differ in their CO tolerance, specific gas uptake rates and specific productivities. In the “wild” strains found in nature, very little ethanol production is noted. Strains of C. ljungdahlii operate ideally at 37° C., and typically produce an ethanol to acetyl (i.e. which refers to both free or molecular acetic acid and acetate salts) product ratio of about 1:20 (1 part ethanol per 20 parts acetyl) in the “wild” state. Ethanol concentrations are typically only 1-2 g/L. While this ability to produce ethanol is of interest, because of low ethanol productivity the “wild” bacteria cannot be used to economically produce ethanol on a commercial basis with minor nutrient manipulation the above-mentioned C. ljungdahlii strains have been used to produce ethanol and acetyl with a product ratio of 1:1 (equal parts ethanol and acetyl), but the ethanol concentration is less than 10 g/L, a level that results in low productivity, below 10 g/L·day. In addition culture stability is an issue, primarily due to the relatively high (8-10 g/L) concentration of acetyl (2.5-3 g/L molecular acetic acid) in combination with the presence of ethanol. Furthermore, as the gas rate is increased in an effort to produce more ethanol, the culture is inhibited, first by molecular acetic acid and then by CO. As a result, the culture becomes unstable and fails to uptake gas and produce additional product. Further, early work by the inventors showed difficulty in producing more than a 2:1 ratio of ethanol to acetyl in a steady state operation. See, e.g., Klasson et al., 1990 Applied Biochemistry and Biotechnology, Proceedings of the 11th Symposium on Biotechnology for Fuels and Chemicals, 24/25: 857; Phillips et al., 1993 Applied Biochemistry and Biotechnology, Proceedings of the 14th Symposium on Biotechnology for Fuels and Chemicals, 39/40: 559, among others.
A large number of documents describe the use of anaerobic bacteria, other than C. ljungdahlii, in the fermentation of sugars that do not consume CO, CO2 and H2 to produce solvents. In an attempt to provide high yields of ethanol, a variety of parameters have been altered which include: nutrient types, microorganism, specific addition of reducing agents, pH variations, and the addition of exogenous gases. See, e.g., Rothstein et al, 1986 J. Bacteriol., 165 (1):319-320; Lovitt et al, 1988 J. Bacteriol., 170 (6):2809; Taherzadeh et al, 1996 Appl. Microbial. Biotechnol., 46:176.
There remains a need in the art of the handling of industrial gaseous substrates, the ability to extract valuable commodities from such gases, particularly waste gases, such as H2, CO and CO2. There is a need to enhance the production of ethanol relative to the production of the other products normally generated by the fermentation of such gases by acetogenic bacteria.